Theodore Marghitu

BIOL 1009 Lab

Group 47: Mariam Ramoni, Rebecca Kinyon, Feven Ayana, Briana Odell, Savanna Briggs
David Begelman
Lab reflection 2
Hypothesis, Experimental Design and Data collection

Introduction:
The positive effects of sodium bicarbonate of bacteria such as Yersinia enterocolita have
been established for a long time (1), but recent media coverage as well as scientific journals has
shown that baking soda (or sodium bicarbonate) also has a positive impact on the health of the
oral cavity by fighting against bacteria such as Lactobacillus or S. mutans, which contribute to
the formation of caries, especially for children (2). Therefore, our goal was to verify these
claims. Our hypothesis was that baking soda has a positive impact on oral cavity health by
eliminating bacteria such as Lactobacillus and S. mutans. If the hypothesis were true, then the
use of baking soda would create an area of inhibition in an agar plate containing bacteria, and
this area of inhibition will increase if the concentration of baking soda is increasing.
Experimental:
The experiment was begun with the preparation of the bacteria. Since the oral cavity was
identified as the place where S. mutans and Lactobacillus could be found, each member of the
group chewed on paraffin wax, and then spit saliva into a 30mL cup. Then, after the paraffin wax
was discarded, 50 μL of saliva was extracted from each cup with a micropipette and was placed
on top of an agar plate using a sterile cell spreader. For each member, three agar plates were
prepared: one which contained nutrients for Lactobacillus, one which contained nutrients for S.
mutans, and one which contained nutrients for both. Then, the edges of the agar plates were
wrapped with parafilm and stored upside down, then placed in an incubator at 37 degrees Celsius
for two days, and then exposed to a temperature of 4 degrees Celsius.
A catalase test was performed in order to make sure that the bacteria did not transform
baking soda into species which did not affect it. In order to perform this test, the colony was
selected to perform catalase test, then a toothpick was used to remove colony from media, and
then the colony was placed on a slide and then 1-2 drops of an aqueous solution of hydrogen
peroxide was added to the colony. Any formation of bubbles was noted down.
The colony morphology of the bacteria was analyzed by looking at the form, elevation,
margin, surface, opacity, pigmentation, and color of the colony. Then, in order to analyze the
morphology of the individual bacteria, a drop of phosphate buffered saline was placed on a
microscope slide, and the colony was placed onto this slide. The slide was then observed under a
compound microscope.
 In order to verify the existence of S. mutans and Lactobacillus in the colony, a
polymerase chain reaction (PCR) was performed. The S. Mutans plate was touched using a
toothpick, and then the toothpick was swirled in 10 uL PCR, which included Taq DNA
polymerase, sterile water, buffer, MgCl2, dNTPs, and primer sets. The sample was placed into a
thermal cycles, and the sequences of both the control 16S rRNA and the 16S rRNA S. mutans
were recorded. The DNA that was amplified was identified using gel electrophoresis. In order to
do that, 1 uL of 10X loading dye was added to sample of 10 uL PCR product, and 5uL of
molecular weight marker was loaded to the well on the left lane of the gel in order to confirm the
size of the PCR product. 10uL of samples was loaded into well of the next lane, and after loading
the samples, the gel box was started up. The cords were connected, and the gel box was run at
150 volts for 40-45 minutes. The gel was then removed with UV transparent gel tray, and a
picture of the gel was taken by illuminating it on the UV transilluminator.
After the identity of the bacteria in the culture was validated, the culture sample was
prepared. In order to do that, the plates were labeled with name, sample type, treatment,
section/date, and agar type. 12 plates were prepared, 6 with BHI for Lactobacillus and 6 with C-
CAN for S. mutans using 100uL of sample, which was distributed using a sterile T-shaped
spreader. Sterile forceps were used to remove discs from dispenser. Then, 4 of the 6 S. Mutans
plates were treated with 100uL of 25%, 50%, 75%, and 100% aqueous solution of baking soda.
Then, one of the remaining two was treated with water (as a negative control), and one with a
commercial antibiotic (as a positive control). Here, the independent variable was the
concentration of baking soda that was used to treat the sample. For Lactobacillus, the procedure
was identical. The samples were then wrapped and stored in a 37 degrees Celsius incubator for
48 hours with disc facing upwards.
The zone of inhibition (the diameter of the clearing zone) (the dependent variable) was
measured on each agar plate. Using this information, the resistance of the bacterial species to
baking soda was determined. Moreover, the influence of concentration of baking soda on the
area of inhibition was determined.

Data:
The catalase test was negative, since there was no formation of bubbles when hydrogen
peroxide was added to the bacterial sample.
The microscopic morphology of Lactobacillus was determined to be long, slender bacilli
which were non-sporing, meaning that they did not create spores. The morphology of S. mutans
was determined to be cocci, 0.5-0.75μm in diameter, grouped in medium-long non-motile chains.
The colony morphology for the two types of bacteria was similar: small punctiform opaque white
colonies.
Using PCR and gel electrophoresis, S. mutans and Lactobacillus were both identified as
being part of the bacterial colony.
The following PCR was found:
 Fig.1: PCRs found for the two types of bacteria
The following values of area of inhibition were found:

Trial 1 Trial 2 Trial 3 Trial 4

Water 0.5 0.5 0.5 0.5
25% 1.1 0.6 2 0.6
50% 1.5 1 3 1.2
100% 1.8 3.5 4.5 1.1
Bleach 2 1.5 1.5 1.4

Fig. 2: Table expressing the different values of the diameter of the area of inhibition for S.
mutans (in cm) using different concentrations of baking soda, as well as water and bleach

Trial 1 Trial 2 Trial 3 Trial 4

0% 0.5 0.5 0.5 0.5
25% 0.6 0.5 1.5 0.8
50% 0.7 0.7 1.2 1.2
100% 1 2.2 3 1.7
Bleach 1.7 2 2.2 3

Fig 3: Table grouping the different values of the diameter of the area of inhibition for
Lactobacilli (in cm) using different concentrations of baking soda, as well as water and bleach
Note: for all these values, the diameter of the disc which was dipped in the solution was
also taken into account. The diameter of this disc is equal to 0.5cm.
Using the data from the previous table, the following graph was created:
 Fig 4: Graph showing the average diameter of the area of inhibition for the different solutions
used, as well as the standard error of measure
Discussion:
The catalase test can be used to distinguish between colonies of yeast cells, which are
catalase positive, and our bacteria of interest. If a colony of cells is catalase positive, then it will
produce bubbles. Therefore, since our colony did not produce bubbles, we were able to infer that
our colony did not include yeast. Catalase is an enzyme produced by organisms to help protect
against reactive oxygen species, like hydrogen peroxide. Catalase converts hydrogen peroxide to
water and oxygen, which appears under the form of bubbles. The negative catalase test made us
hopeful that the bacteria that we grew on our plate were the two bacteria of interest.
By looking at the graph, there is a noticeable positive evolution of the average diameter
of area of inhibition as the concentration of the solution is increased for S. mutans. That means
that, the more baking soda there is in the solution, the more bacteria die, so the antibacterial
quality of baking soda is made evident. The group expected such a correlation because of media
coverage of the positive effects of baking soda on the oral cavity.
Moreover, such results seem to be consistent for the two types of bacteria that were used
in this study (since there is also a positive correlation between the concentration of baking soda
and the diameter of the area of inhibition for Lactobacillus), so the group was able to safely
assume that the effects of baking soda were similar for Lactobacillus and for S. mutans. On the
other hand, it is interesting to notice that the effect is proportionally smaller for Lactobacillus
than for S. mutans.
The null value of the diameter of the area of inhibition for both types of bacteria for the
negative control helped the group see that water (and, therefore, the substance in which the
baking soda was dissolved) did not have any impact on the bacteria, and validates the hypothesis
that baking soda helps fight S. mutans and Lactobacillus in the oral cavity. The positive value for
the positive control showed that the two types of bacteria were not antibiotic-resistant, therefore
emphasizing the antibacterial quality of baking soda. The fact that the value for the positive
control was higher than for the highest concentration of baking soda used shows that the
antibiotic was more efficient at killing the bacteria than baking soda, but such result is normal
considering the fact that antibiotics are made to destroy bacteria.
However, the average for the area of inhibition for the 100% solution of baking soda was
higher than the average area of inhibition for bleach, which is a result that the group did not
expect, since bleach’s purpose is to remove bacteria. It should also be noted that the standard
error of measure for the 100% solution was higher than for bleach, so the results are not very
consistent. Therefore, bleach might still be a more reliable solution for the destruction of
Lactobacillus and S. mutans.
Potential sources of error include the differences in the bacteria belonging in the bacteria
culture, for instance between those living on the margin of the colony as opposed to the center of
the colony. Moreover, in the bacteria cultures, there were multiple species growing in the agar,
which might have intervened with the way the diameter was measured. The inhibition area was
often not circular, so it was sometimes difficult to accurately measure the diameter of the area of
inhibition.

Conclusion:
As a conclusion, I can say that baking soda has indeed an antibacterial effect on S.
mutans and Lactobacillus, which increases with the concentration of baking soda used, so the
hypothesis was validated by the experiment. Although the average value for the 100% solution
of baking soda was higher than for bleach, a conclusion could not be reached about the
comparison between the 100% solution and the bleach solution. Moreover, the effects of the
gastric system should also be taken into account if the effects of baking soda were to be used in
an oral antibiotic (3), as well as the capacity of skin to absorb it if baking soda were to be used in
a cream antibiotic (4). However, its positive effects can be used as an accompaniment to teeth
brushing in order to combat bacteria in the oral cavity, since there are no external actors which
may negatively impact the baking soda. Further research should repeat the experiment with more
concentrated solutions of baking soda and compare these values with the values for bleach, in
order to

Works cited:
(1) Karapinar M, Gönül ŞA. Effects of sodium bicarbonate, vinegar, acetic and citric acids
on growth and survival of Yersinia enterocolitica. International Journal of Food
Microbiology. 1992;16(4):343-347. doi:10.1016/0168-1605(92)90036-3.
(2) Palmer C, Kent R, Loo C, Hughes C, Tanner A. Food and Beverage Consumption
Patterns and S. Mutans in Severe Early Childhood Caries. Journal of the American
Dietetic Association. 2010;110(9). doi:10.1016/j.jada.2010.06.294.
(3) Cole JT, Argenzio RA, Eisemann JH. Physiological responses in swine treated with water
containing sodium bicarbonate as a prophylactic for gastric ulcers12. Journal of Animal
Science. 2004;82(9):2757-2763. doi:10.2527/2004.8292757x.
(4) Whiteley S. Sodium bicarbonate attenuates pain on skin infiltration with lidocaine, with
or without epinephrine. Annals of Emergency Medicine. 1987;16(10):1186.
doi:10.1016/s0196-0644(87)80496-1.